US20150359912A1 - J-Aggregate Forming Nanoparticle - Google Patents

J-Aggregate Forming Nanoparticle Download PDF

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US20150359912A1
US20150359912A1 US14/764,403 US201414764403A US2015359912A1 US 20150359912 A1 US20150359912 A1 US 20150359912A1 US 201414764403 A US201414764403 A US 201414764403A US 2015359912 A1 US2015359912 A1 US 2015359912A1
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nanovesicle
temperature
phospholipid
target site
glycero
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Gang Zheng
Elizabeth Huynh
Kenneth Ng
Mojdeh Shakiba
Robert Weersink
Brian Wilson
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University Health Network
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0036—Porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent

Definitions

  • This application relates to nanoparticles and preferably, nanoparticles that J-type aggregates.
  • the application also relates to nanoparticles useful for fluoresence or photo-acoustic imaging or temperature monitoring.
  • Photoacoustic imaging is a novel imaging technique which utilizes the photoacoustic effect as reported by Alexander Graham Bell over 100 years ago (Bell, 1880). This technique, advanced by Kruger (Kruger, 1994; Kruger et al., 1995), Oraevsky (Oraevsky et al., 1997) and Wang (Wang, 2009; Wang and Hu, 2012; Wang et al., 2003) allows for cross-sectional imaging of biological tissues at depths rivaling existing optical techniques.
  • the principles at work in PAT involve the excitation of intrinsic or extrinsic absorbers using a non-ionizing pulsed laser source.
  • Non-radiative relaxation of the excited absorber by vibrational relaxation leads to the generation of acoustic waves which are then detected by an ultrasound transducer.
  • a 3-dimensional image can be generated.
  • PAI is a relatively inexpensive technique and has potential to synergize with other therapies and imaging modalities (i.e. high-intensity frequency ultrasound, photothermal therapy).
  • imaging modalities i.e. high-intensity frequency ultrasound, photothermal therapy.
  • intrinsic PAI has been actively investigated as a modality for measuring temperature changes as a result of focal thermal therapy in cancer (Chitnis et al., 2009; Shah et al., 2008).
  • the principle of the technique involves the fact that the measured photoacoustic signal amplitudes depend on the temperature of the source object and the signal amplitudes can be used to monitor the temperature (Pramanik and Wang, 2009).
  • the photoacoustic signal depends on many factors such as the level of coagulation, blood concentration and spectral sensitivity. These factors are in turn affected by biological factors such as the degree of tumor vascularization and tumor size (Esenaliev et al., 1999).
  • a highly sensitive, temperature-dependent PAI contrast agent in which the photoacoustic signal generated will not be sensitive to other uncontrolled and unknown environmental factors.
  • Exogenous probes tested in conjunction with PAT include small-molecule dyes and metallic nanoparticles; such as, nanoshells, nanorods, nanocages and carbon nanotubes.
  • the large absorption cross-section of metallic nanoparticles in the near-infrared region of the electromagnetic spectrum makes these agents especially suitable for PAI.
  • J-type aggregates also known as J-aggregates, are formed through edge-to-edge packing of the dye molecules and results in narrowing, red-shifting and enhancement of the absorption band.
  • Other properties characteristic of J-aggregation include: a decreased Stokes shift and enhanced fluorescence. These optical properties can be explained by the interaction between Frenkel excitons; electron-hole pairs localized on individual molecules (Knoester, 2003). The shape of the absorption band is affected by the degree of coupling between dyes molecules based upon their intermolecular orientation.
  • J-aggregation is heavily influenced by temperature.
  • excitons in certain J-aggregates have been found to be delocalized over 1 ⁇ 10 7 molecules (Scheblykin, 2012). This is in contrast to the calculated 1000 molecules at room temperature (Scheblykin, 2012).
  • a nanovesicle having a bilayer comprising (i) a saturated first phospholipid and (ii) no more than about 15 molar % of a second phospholipid covalently conjugated to a J-aggregate forming dye.
  • a nanovesicle having a bilayer comprising (i) a saturated first phospholipid and (ii) a second phospholipid covalently conjugated to a J-aggregate forming dye, wherein the dye does not comprise a porphyrin moeity.
  • a method of monitoring temperature at a target site comprising: providing the nanovesicle of any one of claims 1 - 20 at the target site, and monitoring absorbance at the target site; wherein a blue shift in absorbance is indicative of temperature at the target site being higher than a predetermined temperature, the predetermined temperature corresponding to a transition temperature of the saturated first phospholipid, and wherein a red shift in absorbance is indicative of temperature at the target site being lower than the predetermined temperature.
  • a method of monitoring temperature at a target site comprising: providing the nanovesicle of any one of claims 1 - 20 at the target site, and monitoring a photoacoustic signal at the target site; wherein a lack of a photoacoustic signal is indicative of temperature at the target site being higher than a predetermined temperature, the predetermined temperature corresponding to a transition temperature of the saturated first phospholipid, and wherein a presence of a photoacoustic signal is indicative of temperature at the target site being lower than the predetermined temperature.
  • a method of monitoring temperature at a target site comprising: providing the nanovesicle of any one of claims 1 - 20 at the target site, and monitoring a fluorescence signal at the target site; wherein a presence of a blue shifted fluorescence signal is indicative of temperature at the target site being higher than a predetermined temperature, the predetermined temperature corresponding to a transition temperature of the saturated first phospholipid, and wherein a presence of a red-shifted fluorescence signal is indicative of temperature at the target site being lower than the predetermined temperature.
  • FIG. 1 shows UV-absorption spectra of Bchl-lipid or Bchl-acid in various lipid environments at 4° C. and 37° C.
  • A Absorption spectra of 5% Bchl-lipid in the presence of various phospholipids with either 0 or 1 unsaturated bonds at 4° C.
  • B Absorption spectra of 5% Bchl-acid in the presence of various phospholipids with either 0 or 1 unsaturated bonds at 37° C.
  • C Absorption spectra of 5% Bchl-lipid in the presence of phospholipids with 0 or 1 unsaturated bonds at 37° C.
  • D Absorption spectra of 5% Bchl-acid in the presence of phospholipids with 0 or 1 unsaturated bonds at 37° C.
  • FIG. 2 shows absorption spectra of varying % mol Bchl-lipid in a saturated lipid environment (with 5% DPPE-PEG2000), showing J-aggregation in formulations containing 5%-50% Bchl-lipid.
  • FIG. 3 shows structural characterization of J-nanoparticles
  • A Negative staining transmission electron micrograph of 15% Bchl-lipid J-nanoparticles and (B) corresponding dynamic light scattering trace.
  • FIG. 5 shows (A) Photoacoustic image of gel phantom containing Bchl-lipid vesicles in either a DPPC or POPC environment at two wavelengths of interest. When samples are treated with detergent (0.5% Triton X-100) to disrupt the structure, the photoacoustic signal disappears. (B) Corresponding photoacoustic spectra of the samples in A with UV/Vis spectra for comparison.
  • FIG. 6 shows (A) Temperature melt curve of JNPs prepared with 14-carbon (DMPC), 16-carbon (DPPC), 17-carbon (DHPC), 18-carbon (DSPC) and 19-carbon (DNPC). PA signal was monitored at 824 nm as samples were heated in a waterbath.
  • B UV-Visible absorption melt curve of JNPs.
  • C UV-Visible absorption melt curve of 15% Bchl-lipid DPPC JNPs showing the reversibility of the JNP's 824 nm absorption peak over multiple heat-cool cycles.
  • D Reversibility of 15% Bchl-lipid DPPC JNPs over 5 cycles. Temperature was raised and cooled during each cycle and the signal at 824 nm (green) and 750 nm (red) were recorded. Image of each sample tube during consecutive heat-cool cycles.
  • FIG. 7 shows temperature response of DPPC JNP loaded into gel phantom during heating
  • A PAI of polyacrylamide gel at various times during heating. PA signal at 750 nm (red) and 824 nm (green).
  • B Correlation between thermal front (>41° C.) determined from IR and PA.
  • Image panel on the right shows representative images of the tumor (red scatterplot) at 40° C., 45° C., and 50° C. with top panels showing the ultrasound image (grayscale), blood signal (red; 680 nm-850 nm) and wavelength corresponding to JNPs (green; 824 nm-850 nm). The bottom panels show the 824-850 nm signal alone for clarity.
  • (B) PA imaging of tumors (n 4) injected with 130 uM JNP (intratumoral; 100 uL) and the influence of heating on PA signal.
  • Image panel on the right shows representative images of the tumor (red scatterplot) at 40° C., 45° C., and 50° C. with top panels showing the ultrasound image (grayscale), blood signal (red; 680 nm-850 nm) and wavelength corresponding to JNPs (green; 824 nm-850 nm). The bottom panels show the 824-850 nm signal alone for clarity.
  • Image panel on the right shows representative images of the tumor (red scatterplot) at 40° C., 45° C., and 50° C. with top panels showing the ultrasound image (grayscale), blood signal (red; 680 nm-850 nm) and ICG signal (blue; 810-850 nm). The bottom panels show the 810-850 nm signal alone for clarity.
  • FIG. 9 is a schematic of hypothesized J-nanoparticle structure below and above transition temperature. Below the transition temperature, Bchl-lipid dyes form J-aggregates with red shift absorption. Above the transition temperature, fluidity in the vesicle membrane inhibits J-aggregation, leading to a recovery of the monomer absorption and a decrease in aggregate absorption.
  • FIG. 10 shows transmission electron microscope images of JNP ranging from 5-50% Bchl-lipid content. An increase in Bchl-lipid % beyond 15% led to changes in the vesicle morphology. Scale bar represents 500 nm
  • FIG. 11 shows (A) Absorption spectrum of IRDye QC-1 showing the similar absorbance values at 750 nm and 824 nm. (B) Photoacoustic spectrum showing the similarity of the photoacoustic signal under 750 nm and 824 nm lasing wavelengths.
  • FIG. 12 shows the difference of internal tumor temperature versus bath temperature during heating experiment.
  • Tissue thermocouple was inserted 2 mm into tumor. Heat rate and water bath mixing velocity was matched to that of experiments in FIG. 4 . Temperature differential as measured in each animal during the course of heating. Each bar represents average ⁇ standard deviation of each datapoint in heating trace.
  • J-aggregates can be induced in ordered environments such as in polymer films (Zakharova and Chibisov, 2009), DNA (Kawabe and Kato, 2011) phospholipid membranes (Mo and Yip, 2009) and inorganic nanoparticles (Fofang et al., 2011; Walker et al., 2009).
  • J-aggregation of pseudoisocyanine dyes within a structured phospholipid monolayer can be altered depending on the transition temperature of the host lipid (Mo and Yip, 2009).
  • a nanovesicle having a bilayer comprising (i) a saturated first phospholipid and (ii) no more than about 15 molar % of a second phospholipid covalently conjugated to a J-aggregate forming dye.
  • phospholipid is a lipid having a hydrophilic head group having a phosphate group and hydrophobic lipid tail.
  • the dye is selected from the group consisting of pseudoisocyanine, merocyanine, bis(2,4,6-trihydroxyphenyl)squaraine, Zn-chlorin, tetrtakis(4-sulfonatophenyl)-porphyrin, bacteriochlorin, antimony(III)-phthalocyanine, copper phthalocyanine and perylene bismide, Hypericin, subphtalocyanine, preferably bacteriochlorin.
  • the second phospholipid covalently conjugated to the J-aggregate forming dye is bacteriochlorophyll-lipid.
  • a nanovesicle having a bilayer comprising (i) a saturated first phospholipid and (ii) a second phospholipid covalently conjugated to a J-aggregate forming dye, wherein the dye does not comprise a porphyrin moeity.
  • the dye is selected from the group consisting of pseudoisocyanine, merocyanine, bis(2,4,6-trihydroxyphenyl)squaraine, Zn-chlorin, antimony(III)-phthalocyanine, copper phthalocyanine and perylene bismide, preferably bacteriochlorophyll.
  • the second phospholipid is present in the bilayer in an amount of between 0.01-15 molar %.
  • the second phospholipid is present in the bilayer in an amount of between 2-13 molar %.
  • the second phospholipid is present in the bilayer in an amount of about 5 molar %.
  • the second phospholipid is present in the bilayer in an amount of about 10 molar %.
  • the second phospholipid is present in the bilayer in an amount of about 15 molar %.
  • the second phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanoloamine, phosphatidylserine, phosphatidylinositol, lyso-phosphatidylcholine, lyso-phosphatidylethanoloamine, lyso-phosphatidylserine and lyso-phosphatidylinositol.
  • the second phospholipid comprises an acyl side chain of 12 to 22 carbons.
  • the dye is conjugated to the glycerol group on the second phospholipid by a carbon chain linker of 0 to 20 carbons.
  • the saturated first phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidic acid, phosphatidylglycerols and combinations thereof.
  • the saturated first phospholipid is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diarachidoyl-sn-glycero-3-phosphatidylcholine (DAPC), 1,2-dilignoceroyl-sn-glycer
  • the nanovesicle further comprises PEG-lipid. In some embodiments, the nanovesicle further comprises DPPE-PEG2000. In some embodiments, the nanovesicle further comprises DSPE-PEG2000. In some embodiments, the PEG or PEG-lipid is present in an amount of about 5 molar %.
  • the nanovesicle is substantially spherical and about 110 nm in diameter.
  • a method of monitoring temperature at a target site comprising: providing the nanovesicle of any one of claims 1 - 20 at the target site, and monitoring absorbance at the target site; wherein a blue shift in absorbance is indicative of temperature at the target site being higher than a predetermined temperature, the predetermined temperature corresponding to a transition temperature of the saturated first phospholipid, and wherein a red shift in absorbance is indicative of temperature at the target site being lower than the predetermined temperature.
  • a method of monitoring temperature at a target site comprising: providing the nanovesicle of any one of claims 1 - 20 at the target site, and monitoring a photoacoustic signal at the target site; wherein a lack of a photoacoustic signal is indicative of temperature at the target site being higher than a predetermined temperature, the predetermined temperature corresponding to a transition temperature of the saturated first phospholipid, and wherein a presence of a photoacoustic signal is indicative of temperature at the target site being lower than the predetermined temperature.
  • a method of monitoring temperature at a target site comprising: providing the nanovesicle of any one of claims 1 - 20 at the target site, and monitoring a fluorescence signal at the target site; wherein a presence of a blue shifted fluorescence signal is indicative of temperature at the target site being higher than a predetermined temperature, the predetermined temperature corresponding to a transition temperature of the saturated first phospholipid, and wherein a presence of a red-shifted fluorescence signal is indicative of temperature at the target site being lower than the predetermined temperature.
  • phospholipids were purchased from Avanti Polar Lipids Inc. (Alabaster, Ala.) and reconstituted with chloroform prior to utilization. Bacteriochlorophyll-conjugated lipid was synthesized as previously reported (Lovell et al., 2011). Polyethylene tubing with (1.09 mm internal diameter) was purchased from Becton Dickinson and Company (Sparks, Md.) and was thoroughly washed with ethanol before use. Agarose was purchased from BioRad (Mississauga, ON), while fetal bovine serum was purchased from Wisent (St. Bruno, QC). Extruder drain discs and polycarbonate membranes were purchased from Whatman (Piscataway, N.J.)
  • JNPs were made by the lipid extrusion technique as previously described. Briefly, Bchl-lipid, PEG2000-DPPE and host lipids dissolved in chloroform were transferred to borosilicate glass tubes and dried by N 2 to form a thin film (Table 1-1). For serum stability studies, cholesterol (40 mol %) was added to the formulation. This film was then transferred to a vacuum desiccator and dried for an additional 30 min to ensure complete solvent removal. Films were hydrated with PBS and subjected to 5 freeze-thaw cycles and extruded through two 100 nm polycarbonate membranes using a hand extruder or a high pressure extruder set to a temperature of 65° C.
  • the UV/Vis absorption ratio of J-nanoparticles was measured in PBS using a Varian Cary 50 UV-visible spectrophotometer (company and country). This measurement was divided by the number of moles of Bchl-lipid (37 000M ⁇ 1 cm ⁇ 1 ; 1100 MW) in the solution to estimate the molar extinction coefficient of the aggregated molecule.
  • Transmission electron microscopy was carried out on a Hitachi H-7000 electron microscope with an acceleration voltage of 75 kV. Ten microliters of sample was applied to a glow discharged 200-mesh copper-coated grid. The sample was washed with ddH 2 O and stained with 2% uranyl acetate.
  • JNPs were incubated with 0% and 50% fetal bovine serum at 37° C. over 48 hours.
  • Time points measured include: 0, 0.5, 1, 6, 24 and 48 hr time points.
  • sample was withdrawn from the incubation tube, transferred to 96-wellplate and the absorbance measured at 824 nm.
  • Photoacoustic imaging was performed using a Vevo 2100 LAZR photoacoustic imaging system (Visualsonics, Toronto, ON) equipped with a 21 MHz-centered transducer and a flashlamp pumped 20 Hz Q-switched Nd-YAG laser, tunable from 680-970 nm with a 1 nm step size.
  • the gel phantoms were prepared by pouring 60 mL a boiling 1% agarose solution into a 10 cm Petri dish. Once slightly cooled, an electrophoresis gel comb was placed in the gel and allowed to solidify. The comb was then removed and the wells were filled with the sample mixed with agarose (0.5% final).
  • Photoacoustic imaging was performed using a Vevo 2100 LAZR photoacoustic imaging system (Fujifilm, Toronto, ON) equipped with a 21 MHz-centered transducer and a flashlamp pumped 20 Hz Q-switched Nd-YAG laser, tunable from 680-970 nm with a 2 nm step size.
  • Photoacoustic-temperature profiles were collected in a custom-built heating apparatus comprised of 5 polyethylene tubing fixed within a plastic holder. The plastic tubing and holder was submerged in a glass beaker filled with degassed water and a stir bar. Tubes in the heating apparatus were loaded with JNPs prepared with host phospholipids of various acyl chain lengths.
  • the photoacoustic transducer was placed such that the ultrasound array captured an image slice through each tube.
  • the temperature in the bath was increased from 25-60° C. using a hot plate while being monitored using a thermocouple placed in the same depth of water as the plastic tubing.
  • Polyacrylamide photoacoustic hydrogel phantoms were prepared using the method described by Choi and colleagues with modification. Briefly, 59.06 mL ddH2O, 30 mL of 30% (w/v) 19:1 acrylamide and 10 mL of 1M Tris buffer (pH 8) were combined in an Erlenmeyer flask and degassed under vacuum for 15 min. Ammonium persulfate (APS; 10% w/v) and N,N,N′,N′-tetramethylethylenediamine (TEMED) were added to the monomer solution such that the final concentration was 0.84% and 0.2%, respectively.
  • APS Ammonium persulfate
  • TEMED N,N,N′,N′-tetramethylethylenediamine
  • Polymerizing solution was rapidly poured into a custom built rectangular gel mold and comb and allowed to polymerize for 1 hr.
  • the monomer solution was prepared once again, however a volume of the ddH2O was replaced with a solution of JNP such that the final JNP concentration was 30 ⁇ M.
  • the outer gel was polymerized, the comb was removed and the empty space was filled with the newly prepared JNP gel solution. Gels were used immediately after polymerization.
  • Photoacoustic imaging was performed using a Vevo 2100 LAZR photoacoustic imaging system (Fujifilm, Toronto, ON) equipped with a flashlamp pumped 20 Hz Q-switched Nd-YAG laser, tunable from 680-970 nm with a 2 nm step size.
  • Hydrogel phantoms were placed on a resistive heating element (20V; 25 cm2; McMaster-Carr; cat#35475K263) to provide heat to the JNP filled gel.
  • PA images were collected on the gel phantom during the experiment by aligning a 21 MHz transducer array parallel to the direction of heating and scanning across the gel to generate a 3D image of the gel. The excitation wavelength was alternated between 750 nm and 824 nm during the scan. While the gel images were scanning, thermographic images were captured using an IR camera placed perpendicular to the direction of heating.
  • Thermographic images were analyzed using the MikrospecTM 4.0 imaging software. All other analysis was conducted using ImageJ. The thermal front exceeding 41° C. in the hydrogel phantom was measured and compared with the thermal front determined from the decrease in signal intensity at 824 nm. The data was fit using linear least squares regression through the origin.
  • KB cells were cultured in Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum. Immediately prior to tumor inoculation, KB cells were trypsinized and washed 3 times with phosphate buffered saline. The concentration of cells was adjusted to 2 ⁇ 10 7 cells/mL and kept on ice throughout the experiment. Animals were anaesthetized with a gaseous mixture of isofluorane and oxygen. Once induction of anaesthesia was complete, the hind flank of each animal was inoculated with 2 ⁇ 10 6 cells.
  • thermocouples were inserted in the waterbath as well as within the KB tumor. The tip of the thermocouple was buried 2 mm below the surface of the tumor. Heating on the tumors were conducted as described above (vide supra). The bath temperature and the tissue temperature were compared and the difference between the two calculated for each temperature point.
  • Applicants fixed the composition of each formulation at 5 mol % Bchl and 95 mol % host lipid.
  • Bchl-acid and Bchl-lipid applicants prepared a series of formulations with a series of lipids with variations in the chemical structure as well as the phase transition temperature (Table 1). Each prepared film was hydrated with PBS and sonicated at 65° C. for 1 hr. Samples were next adjusted to the same Bchl concentration and transferred to a 96-wellplate. Wavelength scans from 700-850 nm were made using a temperature-controlled plate reader. Measurements made of Bchl-lipid containing samples at 4° C.
  • 50 uM of 15% Bchl-lipid JNPs made using a J-aggregating, a non-J-aggregating host lipid were injected into the agarose gel phantom.
  • Photoacoustic images of the agarose phantom cross-sections were collected over wavelength range of 680-850 nm ( FIG. 5B ). Large signal intensities were observed at 824 nm for the JNP prepared with DPPC.
  • JNPs prepared with DPPC 14-carbon were embedded in a polyacrylamide gel phantom which mimics the ultrasound properties of tissue. Gels were heated from one face using a resistive heating element while PA and infrared images were collected at an angle perpendicular to the direction of heating. Scans across the gel surface were captured at various points during heating and reconstructed showing the PA signal at 750 nm and 824 nm ( FIG. 7A ). During the course of heating, the progression of the temperature front could be observed by a wave of diminishing signal at 824 nm.
  • This signal decrease was confirmed to be the disaggregation of the J-aggregate peak, not as movement of the nanoparticle out of the imaging view since an increase in the monomeric PA signal at 750 nm was observed.
  • Animals were intratumorally-injected with 100 ⁇ L of saline, DPPC JNP (130 ⁇ M) or indocyanine green (ICG; 130 ⁇ M) through the intratumoral route and immobilized in a custom-built waterbath.
  • the temperature in the bath was increased from 25-52° C., while a PA transducer collected images at various wavelengths (680 nm; 750 nm; 800 nm; 824 nm; 850 nm). Region of interests were drawn over tumors and the PA signal was plotted versus temperature. In the case of these traces, each value was normalized to max.
  • PAI with its advantages over other optical techniques has garnered attention for its unparalleled signal depth resolution and its ability to image endogenous process by exciting endogenous absorbers.
  • contrast-enhanced PAI is an active field of research, as it can provide additional information into biological processes in healthy and disease states, especially when coupled with an appropriate targeting moiety.
  • nanoparticle-based contrast agents greatly extends the utility of PAI as they can potentially encapsulate large numbers of imaging dyes per nanoparticle (Kim et al., 2007; Lovell et al., 2011) and in the case of metallic nanoparticles, can utilize the nanoscale property of surface plasmon resonance to tune and greatly enhance the absorption coefficient of the nanoparticle.
  • J-aggregation causes a red-shift, narrowing and enhancement of the dyes absorption band.
  • the reversible, weak, intermolecular interactions governing the association of J-aggregating dye molecules provide a unique mechanism which can be harnessed to create sensors responsive to the local environment of the dye.
  • Another objective was to determine whether the temperature dependence of J-aggregation can be used as a mechanism to sense changes in the local environment around JNP.
  • Bchl-lipid By inserting Bchl-lipid into a host membrane with various transition temperatures, applicants hoped to modulate the temperature at which the J-aggregate signal was lost. The most direct to test this was to load the various formulations into a tube phantom in a water bath. The temperature of the bath was varied and the photoacoustic signal was scanned.
  • the signal intensity of the J-band with temperature applicants observed that the signal generally varied with the transition temperature of the lipids tested.
  • DPPC appeared to show the sharpest decrease around its transition temperature.
  • DSPC also showed a temperature-dependent decrease but the drop was not as dramatic as that of DPPC. Further experiments will determine whether this can be improved. It is also noted that the time it took for the PA signal change to occur was on the ⁇ 10 0 seconds timescale.
  • J-aggregation can be used to enhance the photoacoustic signal of J-aggregating organic dyes and that at least in the case of Bchl-lipid, this change represents an improvement, as the spectra becomes red-shifted further into the tissue optical window with a concomitant increase in signal intensity.
  • JNPs can potentially be used in monitoring temperature of various focal thermal therapies as the J-aggregate induced PAI contrast enhancement is temperature dependent. We've shown that JNP can potentially be used to monitor therapeutic hyperthermia (41° C.).

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